Method for dispensing drops of different volumes

ABSTRACT

A method that allows a user to dispense a desired volume of solution from an acoustic dispensing apparatus by allowing the user to select the drop volume to be dispensed. A typical drop volume is in the range of one to twenty-five nanoliters. The method comprises the steps of creating two or more burst curves that give the relationship between liquid level and burst value, using data from the burst curves to create two or more calibration functions, and using data from the calibration functions to create a dispensing data set that is used to set the burst parameter required to dispense the selected drop volume.

BACKGROUND OF THE INVENTION

Acoustic dispensing is a well-known method for dispensing very smallvolumes of liquid, for example in the range of one nanoliter to onemicroliter. Generally, multiple drops (sometimes called droplets) havinga fixed volume are ejected from an acoustic dispensing apparatus toyield the total volume of liquid that is desired. This methodology isused because the acoustic dispensing apparatus must be carefullycalibrated to dispense a specific drop volume, making it time consumingto change the volume of the drop. The inability to freely select thevolume of a drop creates several problems. For example, it limits thefinal dispense volume to multiples of the selected drop volume, and itresults in a longer dispense time if the size of the drop is small.Nonetheless, this is the current state of acoustic dispensing usingtechnology such as that discussed below.

FIG. 1 illustrates an acoustic drop dispensing apparatus 10 known in theprior art. Apparatuses of this type are capable of dispensing drops ofliquids having volumes as small as approximately one hundred picoliters,and are particularly useful in the biotechnology and biopharmaceuticalfields. A representative acoustic drop dispensing apparatus is describedin U.S. Pat. No. 6,863,362 which is incorporated herein by reference.

In the apparatus 10, an acoustic wave emitter 14 (such as apiezoelectric crystal) is in electrical communication with a computer18. During operation the acoustic wave emitter 14 generates an acousticwave or beam 20 that can be propagated through an optional wave channel24. The acoustic wave can be focused by a lens 28 prior to propagatingthrough a coupling medium 32 to optimize the energy of the acoustic waveor beam 20 upon the liquid/air interface (free surface) of a sourceliquid 40. The assembly comprised of the acoustic wave emitter 14, thewave channel 24 and the lens 28 is referred to as an acoustic emitterassembly 29. The acoustic wave 20 is propagated through the couplingmedium 32 after which the wave is transmitted through a source liquidcontainment structure 44 where the wave comes to focus at or near thesurface of the pool of source liquid 40, thereby causing a drop 60 ofthe source liquid 40 to be dispensed from the surface of the pool.

Examples of source liquid containment structures 44 include single andmulti-well wellplates commonly used in molecular biology applications,capillaries (e.g., capillary arrays), and the like. However, othercontainers or structures may be used to hold the liquid 40 to bedispensed or ejected. A typical wellplate comprises a matrix (rows andcolumns) of individual wells 46. Typical commercially availablewellplates have 96, 384, 1536 or 3456 individual wells. The sourceliquid 40 may be contained in some or all of these wells 46 and thecomposition of the source liquid in individual wells may differ fromwell to well (i.e. there can be multiple source liquids 40).Furthermore, the volume of source liquid in the individual wells maydiffer from well to well. The volume of source liquid in an individualwell is derived from the liquid level and well geometry.

Optimally, to dispense one or more drops from one of the individualwells 46, the well 46 must be positioned over the acoustic wave emitter14. To accomplish this, the source fluid containment structure 44 isdetachably affixed to a gripper 49. The gripper 49 is controlled by anactuator mechanism 50 which contains a horizontal actuator 54 for movingthe containment structure 44 in the horizontal (x and y) directions. Avertical actuator 58 moves the acoustic wave emitter 14 and wave channel24 in the vertical (z) direction. The actuator 50 is typically incommunication with computer 18 which controls the movement of thecontainment structure 44 to select a source liquid 40 or to adjustfocusing of the acoustic wave or beam 20 at or near the surface of thesource liquid 40. The computer may have implemented thereon variousalgorithms to adjust the focal position and energy of the acoustic waveemitter as well as control and manage the location of the acoustic waveemitter relative to a source fluid present in or on a source fluidcontainment structure.

Accordingly, the apparatus 10 may be used to cause one or more drops 60of the source liquid 40 to be dispensed from the containment structure44 and towards a target substrate 70, as is described in U.S. Pat. No.6,863,362. The target substrate 70 may be a multi-well wellplate likethe source fluid containment structure 44, or may be some other type ofmedium. Generally, one or more horizontal actuators 59 are provided formoving the target substrate 70 in the horizontal (x and y) directions. Atypical wellplate that could be used as the target substrate 70 may have96, 384, 1536 or 3456 individual target wells 74, or some other numberof target wells. FIG. 2 illustrates the target wells 74 in a wellplateused as the target substrate 70.

In many cases, a piezoelectric transducer is employed as an acousticwave emitter 14. For example, the piezoelectric transducer may comprisea flat thin piezoelectric element, which is constructed between a pairof thin film electrode plates. As is understood by those of skill in theart, when a high frequency and appropriate magnitude voltage is appliedacross the thin film electrode plates of a piezoelectric transducer,radio frequency energy will cause the piezoelectric element to beexcited into a thickness mode oscillation. The resultant oscillation ofthe piezoelectric element generates a slightly diverging acoustic beamof acoustic waves. By directing the wave or beam onto an appropriatelens having a defined radius of curvature (e.g., a spherical lens, orthe like), the acoustic beam can be brought to focus at a desired point.

Generally, a computer sends an analog voltage pulse to the piezoelectrictransducer by an electrical wire 78. The electronics can control themagnitude and duration of the analog voltage pulses, and the frequencyat which the pulses are sent to the piezoelectric transducer. Eachvoltage pulse causes the generation of an acoustic wave from thepiezoelectric transducer, which in turn is propagated through a couplingmedium and into or through the source fluid thereby impinging on thesurface of the source fluid. A series of cycles of acoustic waves andone “off” period after the generation of the acoustic waves(corresponding to an interval between voltage pulses) is referred to asone “burst.”

A problem encountered in using acoustic drop dispensing systems, such asthe apparatus 10, is that it is difficult to precisely control thevolume of the drops dispensed from the apparatus. In large part, this isbecause many parameters associated with the source liquid, such aschemical composition, viscosity, temperature, speed of sound in theliquid, etc., affect the size (volume) of the drop. Furthermore, theliquid level of the source liquid in the well 46 also affects the size(volume) of the drop. Additionally, other factors, such as the geometryof the source well (e.g. well shape, well bottom thickness, etc.) or themanufacturing variability of the acoustic emitter assembly 29, caninfluence the size of the drop. To deal with this problem, the acousticdrop dispensing apparatus 10 needs to be calibrated so that uniform dropvolume can be achieved. A method for calibrating the apparatus 10 isdescribed in U.S. Pat. No. 7,661,289 which is incorporated herein byreference.

As was mentioned previously, the inability to freely select the volumeof a drop to be dispensed limits the final dispense volume to multiplesof the selected drop volume, and results in a longer dispense time ifthe size of the drop is small. What is needed is the ability to selectand dispense drops of any volume within a reasonable range of dropvolumes. This would allow the drop volume to be optimized based on thefinal volume of source solution to be dispensed. In other words, fewerdrops of larger volume could be used to accomplish the dispense volume,and the user could choose the dispense volume that is desired.

SUMMARY OF THE INVENTION

Briefly, the present invention is an acoustic dispensing method thatallows the user to select the final total volume of solution to bedispensed. The method creates the most efficient drop volume calibrationneeded for dispensing by the acoustic dispensing apparatus. The user canalso manually select the drop volume that allows the dispense time to beminimized because the volume of the drops can be chosen to minimize thenumber of drops that need to be dispensed to yield the final totalvolume.

The method comprises the steps of creating two or more burst curves thatgive the relationship between liquid level and burst value, using datafrom the burst curves to create two or more calibration functions, andusing data from the calibration functions to create a dispensing dataset that is used to set the burst parameter required to dispense theselected drop volume. In a typical procedure, the user determines thenumber of drops needed to dispense the desired volume of solution,calculates the required drop volume, uses the liquid level of thesolution to select the burst parameter from the dispensing data set, anddispenses the drops.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a schematic diagram of an acoustic drop dispensing apparatusof the prior art;

FIG. 2 is a top view of a wellplate of the prior art;

FIG. 3 is a flow chart according to the present invention;

FIG. 4 is a plurality of burst curves;

FIG. 5 is a graph of a calibration function according to the presentinvention;

FIG. 6 is a graph of the slopes of a plurality of calibration functionsaccording to the present invention;

FIG. 7 is a graph of the intercepts of a plurality of calibrationfunctions according to the present invention;

FIG. 8 is a cross sectional view of a well in a wellplate;

FIG. 9 is a graph illustrating the drop volume required to dispense arequested volume; and

FIG. 10 is a flow chart illustrating a method of using the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a method that allows a user to select any dropvolume within a specified range for dispensing by the acousticdispensing apparatus 10. Among other things, the ability to select thedrop volume allows the dispense time to be minimized because the volumeof the drops can be chosen to minimize the number of drops needed toyield the total volume that needs to be dispensed. It also gives theuser more freedom in selecting the total volume to be dispensed, becausethe dispensing process is no longer limited to one or two drop sizes.

The ability to select the drop volume is accomplished through aprocedure that creates two or more calibration functions that relatedrop volume to burst over a range of liquid levels in a containmentstructure, such as a well in a wellplate. In the preferred embodiment,the calibration function is generated through a multiple part method,whose endpoint is a dispensing data set allows the burst needed toproduce the desired drop volume to be set. FIG. 3 is a flow chart thatsummarizes the sequence of steps used in generating the dispensing dataset that allows a user to choose any drop volume within a selected rangefor dispensing by the acoustic dispensing apparatus 10.

Step 130 in FIG. 3 illustrates the first step in the procedure forgenerating the calibration functions. In step 130, a series of burstcurves are derived for a range of drop volumes. A burst curve is a plotof liquid level versus burst value for a fixed drop volume. In arepresentative calibration procedure, the data for a plurality of burstcurves is collected for a plurality of drop volumes. For example, FIG. 4illustrates a set of seven burst curves for seven different dropvolumes, such as 2, 4, 6, 7, 8, 9, and 10 nanoliters. In the preferredembodiment, the burst curves are generated as part of the calibrationprocedure used in a commercially available acoustic dispensingapparatus, and are discussed later in this patent application.

In step 136, for a fixed liquid level value (e.g. 3.01 mm), drop volumeversus burst parameter is plotted for the drop volumes used in the firststep 130 (seven drop volumes in this example). In other words, by usingthe burst curves from FIG. 4 and step 130, the burst value for each ofthe drop volumes is extracted from the burst curve at the fixed liquidlevel, and plotted versus drop volume, as is illustrated in FIG. 5. Step142 illustrates that the data from step 136 is fitted to a function,referred to as the calibration function, that can be used to determinethe burst value needed to produce a selected drop volume of any size atthe specified liquid level.

In the preferred embodiment, the function that results from step 142 isthe equation for a straight line relating drop volume to burst value fora given liquid level. Therefore, the slope and intercept of thisfunction can be used to calculate the burst value needed to produce anyselected drop volume at the specified liquid level. The curve fittingprocess can be accomplished by several methods. For example, commercialsoftware like Microsoft's Excel spreadsheet can be used, as can NationalInstruments' LabVIEW math functions software. Alternatively, a curvefitting routine, like a least squares fitting routine, can be customwritten and compiled in a computer language like C++.

FIG. 5 illustrates the graph generated in steps 136 and 142 using thedata from FIG. 4 at the liquid level of 3.01 mm, and shows thecalibration function 194 as a straight line. In other embodiments, thecalibration function may not be a straight line (i.e. the calibrationfunction might be a curve), and a higher degree polynomial would berequired for an acceptable the curve fit.

Step 148 indicates that in the preferred embodiment, a plurality ofcalibration functions is needed for a plurality of liquid levels (e.g.36 calibration curves for 36 liquid levels). The plurality ofcalibration functions is generated by repeating steps 136 and 142 forthe plurality of liquid levels, thereby yielding a plurality ofdifferent calibration functions analogous to the calibration function194 shown in FIG. 5.

Step 154 indicates that each of the plurality of different calibrationfunctions has a slope and intercept (or other coefficients) that can beused to calculate the burst needed to produce any selected drop volumeover the range of liquid levels. A lookup table (called the dispensingdata set) is created in step 154 for storing the slope and intercept andany other relevant information as a function of liquid level.

FIGS. 6 and 7 show the results of Step 154 in graphical form. FIG. 6illustrates the individual values for the slopes of the plurality ofcalibration functions plotted over the range of liquid levels from 0.05to 6.54 millimeters, and FIG. 7 illustrates the individual values forthe intercepts of the plurality of calibration functions plotted overthis range of liquid levels. The data contained in FIGS. 6 and 7 can beused to calculate the burst value needed to produce any selected dropvolume over the range of liquid levels, as is explained later.

In a preferred embodiment, the data in FIG. 4 was generated using a Gen5 ATS acoustic dispenser from EDC Biosystems of Fremont, Calif. The Gen5 ATS acoustic dispenser is similar to the acoustic dispensing apparatus10 shown in FIG. 1, and in the discussion below, the components of theacoustic dispenser 10 shown in FIG. 1 are used to describe the Gen 5 ATSacoustic dispenser.

The seven burst curves shown in FIG. 4 show what burst value must beused in the acoustic dispenser 10 to dispense the specified drop size(volume) over a range of liquid levels. A burst is a series of acousticwaves followed by a period of rest, such as an “off” period after thegeneration of the acoustic waves. The off period corresponds to aninterval between voltage pulses applied to the acoustic wave emitter 14that cause the acoustic waves to be emitted. Therefore, a series ofbursts is proportional to the length of time that an acoustic signal isapplied to a source liquid. In other words, the amount of energy beingapplied to the surface of the liquid is proportional to both thestrength of the acoustic wave and the length of time that those acousticwaves are being applied. In this application, the terms burst, burstvalue, and burst parameter are used interchangeably.

In order to generate a burst curve, the apparatus 10 must be calibratedto determine what burst will yield a specific drop volume at variousliquid levels in the source well. In the preferred embodiment, a methodfor generating burst curves such as the one described in U.S. Pat. No.7,661,289, is used. In other embodiments, other methods could be used.In general, the calibration procedure involves using the apparatus 10 todispense drops of a solution containing a dye, such as a fluorescentdye, into target wells, and then calculating the drop volume that wasdispensed by comparison to a standard having a known concentration ofthe fluorescent dye. This process is repeated for a plurality of liquidlevels, and then the data is processed to yield the burst curve showingwhat burst is required to produce a drop of a given volume as a functionof liquid level (i.e. as a function of the height of the source liquidin a well or some other container).

Once an acceptable burst curve is obtained, it can be further processedto yield a fine tuned burst curve. Fine tuning is done by selecting theburst settings from a burst curve for a particular drop volume, and thenusing the settings to dispense a dye solution, such as fluorescein dyein a DMSO solution, onto a target plate using the apparatus 10. Thefluorescent counts versus liquid level are then plotted and compared tothe fluorescent counts expected based on the selected drop size. Theburst values for each liquid level are then changed until thefluorescent counts are roughly uniform over the range of liquid levels,indicating that a uniform (and accurate) drop size is being dispensed ateach liquid level. A calibration is considered finely tuned when therelative standard deviation is less than 5% along the range of liquidlevels. The mean value of the data is used as the actual drop volumedispensed in the process.

FIG. 8 illustrates that the liquid level “L” of the source liquid 40 inthe source well 46 is the height of the free surface of the liquid 40above the bottom of the well 46. Generally, L is the distance betweenthe lowest part of the meniscus 84 of the liquid 40, and a well bottomsurface 86 of the well 46 that is in contact with the liquid 40.However, other reference points could be used as the liquid level.

FIG. 8 also illustrates that the wellplate 44 has a thickness “T”underneath the well bottom surface 86. The wellplate 44 has a wellplatebottom surface 88. In the preferred embodiment, liquid level is measuredby the acoustic dispenser 10, such as by measuring the time it takes foran acoustic wave to make a round trip from the acoustic wave emitter 14to the surface of the source liquid 40 (i.e. the meniscus 84), calledt1, and subtracting out the time it takes for an acoustic wave to make around trip to the bottom surface 86 of the well, called t2. The liquidlevel (LL) is then calculated using a calculation such as LL=v(t1−t2)/2,where v is the speed of sound in the liquid 40, as is explained in U.S.Pat. No. 7,661,289. However, other methods of measuring liquid levelcould be used.

Referring to FIG. 4, seven finely-tuned burst curves are shown for sevendifferent drop volumes of a 90% DMSO/10% water/100 μM fluoresceinsolution. These curves are labeled 164, 168, 172, 176, 180, 184, and188, and correspond to drop volumes of 2 nanoliters, 4 nl, 6 nl, 7 nl, 8nl, 9 nl, and 10 nl, respectively. In practice, since this calibrationprocedure is implemented in software, the data for the burst curves arestored as a calibration files in electronic memory (usually on a harddisk and in RAM).

A calibration file is created for each drop volume (i.e. sevencalibration files in this example), and each calibration file comprisesa look up table that lists a variety of parameters required to dispensethe given drop volume. These parameters include the drop volume, focus,voltage, and burst for each of thirty-six liquid levels. In thepreferred embodiment, the focus and voltage are held constant, so onlythe burst varies with liquid level. In other embodiments, the focus andvoltage could be varied, and other parameters could be included. Thecalibration files are referred to as burst curve data sets in otherparts of this application.

FIG. 5 utilizes the data shown in FIG. 4, and shows seven burst values,one burst value for each drop volume, plotted against the drop volumefor a single liquid level (e.g. 3.01 mm in this case). In other words,FIG. 5 is generated by going to FIG. 4 and reading the burst value at3.01 mm for each of the seven drop volumes. (In practice, thisinformation would be extracted from the calibration files for the burstcurves). In the preferred embodiment, the data in FIG. 5 are subjectedto a curve fitting process, which in this case yields a straight linereferred to as a calibration function 194. Additionally, in thepreferred embodiment two or more new calibration functions are generatedin the same way that the calibration function 194 was generated, exceptthat a new liquid level (and the corresponding new burst values) is usedto generate each of the new calibration functions. For example, in FIG.6 below, a total of thirty-six calibration functions were generated andsubjected to a curve fitting process.

The equation that results from the curve fitting process for thecalibration function 194 is a linear equation that relates drop volumeto burst value for a given liquid level. Therefore, the slope andintercept of the function 194 can be used to calculate the burst valueneeded to produce any selected drop volume at the specified liquidlevel. A least squares analysis of the data in FIG. 5 yields acoefficient of determination (R²) of 0.9966, indicating a very good fitof the data to the straight line (calibration function 194). In otherembodiments, the calibration function may not be a straight line (i.e.the calibration function might be a curve), and a polynomial having adegree higher than one (e.g. 2-10) would be required for an acceptablethe curve fit.

FIG. 6 shows the slopes for thirty-six calibration functions plottedagainst liquid level. The thirty-six calibration functions weregenerated in the same way that the calibration function in FIG. 5 wasgenerated. Specifically, the burst value for each of the seven dropvolumes in FIG. 4, is plotted against the drop volume for a singleliquid level, calculating the slope and intercept of the resulting line,and then plotting the slope versus the liquid level to yield one of thedata points in FIG. 6. This is repeated thirty-five additional times toyield the results shown in FIG. 6. A curve 200 may be drawn thatconnects all of the thirty-six data points in FIG. 6. FIG. 7 shows theintercepts for the thirty-six calibration functions plotted againstliquid level. A curve 210 may be drawn that connects all of thethirty-six data points in FIG. 7.

The data contained in FIGS. 6 and 7 can be used to calculate the burstvalue needed to produce any selected drop volume over the range ofliquid levels. For example, Equation 1 can be used to calculate therequired burst value:Required burst=(slope)(desired drop vol.)+intercept  (1)where the slope and intercept are obtained from FIGS. 6 and 7, and theuser measures the liquid level and chooses the desired drop volume.

In a preferred embodiment, once a liquid level is measured and a desireddrop volume has been selected, then the defined function for the pointhigher in the liquid level and the next point lower in the liquid levelmay be determined. The value for the actual point is determined byinterpolation to the point measured and the proper burst value isacquired. For example, if the liquid level was measured at the point 202on the curve 200, then the slopes for points 204 and 206 in FIG. 6 wouldbe determined, and the slope for point 202 would be determined byinterpolation between these two burst values. Similarly, the interceptfor the point 212 in FIG. 7 (at the measured liquid level) would bedetermined by interpolation between the points 214 and 216. Theinterpolated values for the slope and intercept are then used inequation one to get the required burst for the new drop volume.

FIGS. 6 and 7 show the results of plotting two coefficients, slope andintercept, for a linear calibration function, such as the calibrationfunction 194 obtained in FIG. 5. However, if the calibration function194 was not a straight line, a higher degree polynomial would berequired to fit the data to a curve. This higher degree polynomial wouldhave additional coefficients that would be plotted in the same mannerthat the slope and intercept in FIGS. 6 and 7 were plotted.

In a preferred embodiment, the present invention is implemented insoftware, so all of the data from the FIGS. 6 and 7 are stored in alookup table in electronic memory. The lookup table lists liquid level,slope, intercept, higher degree coefficients (if any), and any otherdesired information (such as constants) in separate columns.Subsequently, an algorithm extracts the required data from the lookuptable to yield the burst value needed to yield the specified drop volumeat a given liquid level.

A preferred embodiment of the method for using the present invention todispense drops of source fluid 60 having any volume within a definedrange is as follows: In a first step, a first burst curve data set 164is created (e.g. using the apparatus 10) that relates a range of liquidlevels (FIG. 4, x-axis) of a source liquid 40 to a range of burst values(FIG. 4, y-axis) for dispensing one or more drops of the source liquidhaving a first drop volume (e,g, 2 nl), with the burst values beingrelated to a plurality of acoustic waves 20. In FIG. 4, thirty-sixreading at 36 liquid levels were used to create the burst curve 164, soall of these data points are included in the term “first burst curvedata set.” The phrase “burst values being related to a plurality ofacoustic waves” means that burst is a series of acoustic waves followedby a period of rest.

In a second step, a second burst curve data set 188 is also created thatrelates the range of liquid levels to the range of burst values fordispensing one or more drops of the source liquid having a second dropvolume (e.g., 10 nl), where the second drop volume is not equal to thefirst drop volume. More burst curve data sets (i.e., a plurality) couldbe created, such as the seven burst curves shown in FIG. 4, but twoburst curve data sets are the minimum if the calibration function isgoing to be a straight line.

In general, the defined range of drop volumes that can be dispensedusing the present invention is approximately determined by the range ofdrop volumes used to create the burst curve data sets, which is 2 nl to10 nl in this example. However, in other cases other ranges of dropvolumes could be used. Frequently, the properties of the source solutionbeing dispensed will influence the range of drop values selected. Apreferred range of drop values is 1 nl to 25 nl. Additionally, in somecases, the defined range of drop volumes could be expanded outside ofthis range used to create the burst curve data sets, if the accuracy inthe drop volumes produced outside of the range is acceptable.

In a third step, a first calibration function data set 194 that relatesthe first drop volume (2 nl) to a first burst value measured at a firstliquid level (3.01 mm in FIG. 5) in the first burst curve data set, andthat relates the second drop volume (10 nl) to a second burst valuemeasured at the first liquid level in the second burst curve data set.

In a fourth step, a second calibration function data set is created thatrelates the first drop volume to a third burst value measured at asecond liquid level in the first burst curve data set, and that relatesthe second drop volume to a fourth burst value measured at the secondliquid level in the second burst curve data set. Here, the second liquidlevel is a liquid level not equal to the first liquid level. Morecalibration function data sets (i.e., a plurality) could be created,such as the thirty-six calibration function data sets used in FIG. 6,but two calibration function data sets are the minimum that would workin the present invention. The more calibration function data sets thatare created, the better the usefulness of the invention.

In a fifth step, a dispensing data set created from the first and secondcalibration function data sets is used to calculate a first new burstvalue required to dispense one or more drops of the source liquid havinga first new drop volume, where the first new drop volume is differentfrom both the first drop volume and the second drop volume. In apreferred embodiment, the first new burst value is calculated using themethod described previously with respect to the points 202 and 212 inFIGS. 6 and 7, respectively. In the preferred embodiment, the first andsecond calibration function data sets are processed to yield two lines(i.e. two equations for lines), and the slopes and intercepts of thesetwo lines are used in the dispensing data set. In other embodiments, thefirst and second calibration function data sets are processed to yieldtwo polynomials having a degree higher than one, or some other type ofequations that describe more complex curves, and the coefficients fromthese two polynomials, or from the other equations, are used in thedispensing data set.

An important advantage of creating new volume calibrations on the fly isthe ability to create a final dispense volume more efficiently (i.e.faster) by using the largest drop volume possible. In general, the mostefficient method for achieving a final dispense volume (i.e. the totalvolume dispensed by a plurality of drops), is to use the largest dropvolume that can be multiplied by an integer to yield the final dispensevolume. In considering this issue, it should be recognized that minimumresolution for dispensing a drop is one burst. It is known that thereare about 60 bursts per nanoliter of solution. This resolutioncorresponds to less than 2% of a one nanoliter dispense (i.e. 1/60 of ananoliter is approximately 2%).

To illustrate these advantages, FIG. 9 shows the drop volume requiredfor a final dispense volume in the range of 0 to 100 nl, where themaximum drop size is 25 nl and the minimum drop size is 1 nl. The majorpoint in FIG. 9 is that for any volume between 1 nl and 100 nl, amaximum of four drops is required to deliver the final dispense volume.For example, looking at 60 nl along the x-axis in FIG. 9, and reading upuntil the line 234 is intersected, shows that a drop volume of 20 nl isthe required drop size (3×20 nl=60 nl).

The information conveyed by FIG. 9 is important because it takes about30 milliseconds to dispense a drop, so it would take about 2.94 seconds(98×30 msec) to dispense 99 nl of solution as 1 nl drops. In contrast,if 99 nl are dispensed using four drops of 24.75 nl, as can be done withthe present invention, is only 0.09 seconds (3×30 msec). So, the methodof the present invention improves (reduces) the time required todispense a volume of solution, and also improves the resolution of thefinal dispense volume.

FIG. 10 illustrates how a user of the acoustic dispensing apparatus 10would use a preferred embodiment of the present invention to dispense avolume of source fluid. In step 244, the user specifies the maximum dropvolume that can be used. In step 248, the user specifies the volume ofsource fluid that should be dispensed (in a single well). In step 252the number of drops to be used to dispense the total volume from step248 is calculated. In the preferred embodiment, Equation 2 is used forthis, but other equations are acceptable. Additionally, the user canmanually select and/or decide on the number of drops to be dispensed.Total number of drops=integer(total volume/maximum drop volume)+1  (2)In this Equation 2, the one extra drop is used to ensure that themaximum drop volume isn't exceeded.

In step 256, the volume of the individual drops is automaticallycalculated, such as by dividing the volume to be dispensed by the numberof drops. In step 260, the liquid level in the source well isautomatically measured by the apparatus 10. In step 264, based on theliquid level from step 260, the coefficients for a calibration curvedependent on the liquid level are looked up, such as the slope andintercept coefficients from FIGS. 6 and 7. In step 268, the burst forthe required drop volume is automatically calculated from thecoefficients obtained in step 264, such as by using Equation 1: Requiredburst=(slope)(desired drop vol.)+intercept. Finally, in step 272 therequired number of drops having the correct drop volume are dispensed byan acoustic dispensing apparatus, such as the apparatus 10.

Although the present invention has been described in terms of thepresently preferred embodiments, it is to be understood that suchdisclosure is not to be interpreted as limiting. Various alterations andmodifications will no doubt become apparent to those skilled in the artafter having read the above disclosure. Accordingly, it is intended thatthe appended claims be interpreted as covering all alterations andmodifications as fall within the true spirit and scope of the invention.

I claim:
 1. A method for dispensing drops of a source liquid with a dropdispensing apparatus, the method comprising: creating a first burstcurve data set that relates a range of liquid levels of the sourceliquid to a range of burst values for dispensing one or more drops ofthe source liquid having a first drop volume, the range of burst valuesbeing related to a plurality of acoustic waves; creating a second burstcurve data set that relates the range of liquid levels to the range ofburst values for dispensing one or more drops of the source liquidhaving a second drop volume, where the second drop volume is not equalto the first drop volume; creating a first calibration function data setthat relates the first drop volume to a first burst value measured at afirst liquid level in the first burst curve data set, and that relatesthe second drop volume to a second burst value measured at the firstliquid level in the second burst curve data set; creating a secondcalibration function data set that relates the first drop volume to athird burst value measured at a second liquid level in the first burstcurve data set, and that relates the second drop volume to a fourthburst value measured at the second liquid level in the second burstcurve data set; creating a dispensing data set based on a firstcoefficient of a first function corresponding to the first calibrationfunction data set and a second coefficient of a second functioncorresponding to the second calibration function data set; calculating,based on the dispensing data set, a first new burst value required todispense one or more drops of the source liquid having a first new dropvolume, where the first new drop volume is different from both the firstdrop volume and the second drop volume; and dispensing, using the dropdispensing apparatus and based on the first new burst value, one or moredrops of the source liquid having the first new drop volume.
 2. Themethod of claim 1 wherein the first and second drop volumes are in arange of one nanoliter to 25nl.
 3. The method of claim 1 wherein:creating the first calibration function data set comprises generating afirst equation for a first line having a first slope and a firstintercept; and creating the second calibration function data setcomprises generating a second equation for a second line having a secondslope and a second intercept.
 4. The method of claim 3 wherein thedispensing data set comprises the first and second slopes and the firstand second intercepts as a function of liquid level.
 5. The method ofclaim 1 further comprising: creating a plurality of burst curve datasets for a plurality of drop volumes, with each of the plurality ofburst curve data sets relating the range of liquid levels to the rangeof burst values for dispensing one or more drops of the source liquidhaving a uniform drop volume, and with each of the plurality of burstcurve data sets yielding a different uniform drop volume from any otherof the plurality of burst curve data sets, the plurality of burst curvedata sets being in addition to the first and second burst curve datasets for the first and second drop volumes.
 6. The method of claim 5wherein the plurality of drop volumes, and the first and second dropvolumes, are all in a range of 1nl to 25nl.
 7. The method of claim 5further comprising: creating a plurality of calibration function datasets that relate the plurality of drop volumes to a plurality of burstvalues measured at a plurality of liquid levels in the plurality ofburst curve data sets, and where each of the plurality of calibrationdata sets is measured at a different liquid level in the plurality ofliquid levels, the plurality of calibration function data sets being inaddition to the first calibration function data set and the secondcalibration function data set.
 8. The method of claim 7 wherein:creating the plurality of calibration function data sets comprisesgenerating a plurality of equations for a plurality of polynomialshaving a degree higher than one; and creating the first and secondcalibration function data sets comprises generating a first and a secondequation for a first and a second polynomial having degrees higher thanone.
 9. The method of claim 8 wherein the plurality of polynomials, andthe first and second polynomials, are used in creating the dispensingdata set.
 10. The method of claim 1 wherein the one or more drops aredispensed using acoustic energy.
 11. The method of claim 1 wherein thefirst coefficient comprises a slope of the first function and the secondcoefficient comprises a slope of the second function.
 12. The method ofclaim 1 wherein the first coefficient comprises an intercept of thefirst function and the second coefficient comprises an intercept of thesecond function.
 13. The method of claim 1 wherein the first coefficientcomprises a slope of the first function and the second coefficientcomprises a slope of the second function, and wherein the dispensingdata set is further based on: a third coefficient comprising anintercept of the first function; and a fourth coefficient comprising anintercept of the second function.
 14. The method of claim 1 whereincalculating the first new burst value comprises: determining a firstcoefficient of the dispensing data set based on a first new liquidlevel; and calculating the first new burst value based on the firstcoefficient and the first new drop volume.
 15. The method of claim 14wherein determining the first coefficient of the dispensing datacomprises interpolating between the first coefficient of the firstfunction and the second coefficient of the second function.
 16. Themethod of claim 15 wherein the first coefficient of the first functioncomprises a slope of the first function and the second coefficient ofthe second function comprises a slope of the second function.